Cloning, Expression, and Purification of Hyperthermophile α-Amylase from Pyrococcus woesei

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• 作者：Amir Ghasemi^a ; Sobhan Ghafourian^b ; Sedighe Vafaei^c ; Reza Mohebi^b ; Maryam Farzi^c ; Morovat Taherikalani^d ; Nourkhoda Sadeghifard^b ; ^{sadeghifard@gmail.com" class="auth_mail" title="E-mail the corresponding author}
• 关键词：amylase ; expression ; recombinant protein ; thermophile
• 刊名：Osong Public Health and Research Perspectives
• 出版年：2015
• 出版时间：December 2015
• 年：2015
• 卷：6
• 期：6
• 页码：336-340
• 全文大小：525 K

文摘

In an attempt α-amylase gene from Pyrococcus woesei was amplified and cloned into a pTYB2 vector to generate the recombinant plasmid pTY- α-amylase.

Methods

Escherichia coli BL21 used as a host and protein expression was applied using IPTG. SDS-PAGE assay demonstrated the 100 kDa protein. Amylolytic activity of proteins produced by transformed E. coli cells was detected by zymography, and the rate of active α-amylase with and without the intein tag in both soluble conditions and as inclusion bodies solubilized by 4M urea were measured.

Results

Amylolytic activity of ∼185,000 U/L of bacterial culture was observed from the soluble form of the protein using this system.

Conclusion

These results indicate that this expression system was appropriate for the production of thermostable α-amylase.